CHARACTERIZATION OF THE STRUCTURAL REQUIREMENTS FOR A CARBOHYDRATE-BASED ANTICOAGULANT WITH A REDUCED RISK OF INDUCING THE IMMUNOLOGICAL TYPE OF HEPARIN-ASSOCIATED THROMBOCYTOPENIA

HAT is the most frequent drug induced immune-thrombocytopenia. We rece ntly identified multimolecular PF4/heparin complexes as the major anti gen. In order to evaluate the structural requirements for formation of the antigenic complex. we chemically synthesized 13 glucan sulfates a nd used 5 heparin fractions (2.4-4.8 kD) and a synthesized pentasaccha ride, representing the antithrombin III binding sequence of heparin, t o further characterize the HAT antigen. In the presence of glucan sulf ates and heparin, HAT antibodies caused platelet activation typically at low but not at high concentrations, as measured by C-14-5HT release . The concentration range giving the activation pattern depended on th e degree of sulfation (DS) and molecular weight (MW) of the glucan sul fates but not on the type of glycosidic linkage of a polysaccharide. W ith linear glucan sulfates with a chain length of 35 monosaccharides, the critical DS to form the HAT antigen ranged between 0.60 and 1.20. Glycosidic branched glucan sulfates were able to form the HAT antigen at a lower DS and a lower MW than linear glucan sulfates. Platelet act ivation by HAT-antibodies in the presence of linear curdlan sulfate fr actions was dependent on their MW. At a low concentration (0.01 mu M) medium-size fractions (60 kD) caused platelet activation but neither s mall (12 kD) nor large fractions (>150 kD) did. Ar higher concentratio ns (2 mu M) the opposite reaction pattern was observed. In the case of heparin, the optimal chain length for forming the HAT antigen is a he xadecasaccharide (4.8 kD). Antigen generation decreased with larger an d smaller fractions. For 50% platelet activation by HAT antibodies inc reasing concentrations of heparin were necessary using heparins with d ecreasing MW: 0.02 +/- 0.015 mu M (4.8 kD), 0.09 +/- 0.016 mu M (3.0 k D), 0.8 +/- 0.21 mu M (2.4 kD). In the presence of the pentasaccharide . HAT antibodies did not cause platelet activation at any concentratio n tested, nor bound to PF4-pentasaccharide complexes in a PF4 based EL ISA system. We conclude that generation of the HAT antigen is dependen t on the ratio of MW and DS of a glucan sulfate. We conclude from this study that a carbohydrate based anticoagulant with a reduced risk of forming the HAT antigen should be linear with a DS <0.6 or a MW <2.4 k D. These data might be important for the design of new drugs for paren teral anticoagulation.